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Animals 2022, 12, 681. https://doi.org/10.3390/ani12060681 www.mdpi.com/journal/animals

Article

Integrated Taxonomy Revealed Genetic Differences in

Morphologically Similar and Non‐Sympatric Scoliodon

macrorhynchos and S. laticaudus

Kean Chong Lim 1, William T. White 2, Amy Y. H. Then 3,*, Gavin J. P. Naylor 4, Sirachai Arunrugstichai 5

and Kar‐Hoe Loh 1,*

1 Institute of Ocean and Earth Sciences, Universiti Malaya, Kuala Lumpur 50603, Malaysia;

[email protected] 2 CSIRO National Research Collections Australia, Australia National Fish Collection,

Hobart, TAS 7001, Australia; [email protected] 3 Institute of Biological Sciences, Universiti Malaya, Kuala Lumpur 50603, Malaysia 4 Florida Museum of Natural History, Dickinson Hall, Gainesville, FL 32601, USA; [email protected] 5 Aow Thai Marine Ecology Centre, Bangkok 10100, Thailand; [email protected]

* Correspondence: [email protected] (A.Y.H.T.); [email protected] (K.‐H.L.)

Simple Summary: In this study, the species identities of similar‐looking coastal spadenose sharks

from different areas were clarified by adding new molecular markers and more individual body

measurements, including animals from the Malaysian Peninsula that had not been examined pre‐

viously. Collective evidence showed that there are two genetically distinct species that do not over‐

lap in their spatial occurrence. The Malacca Strait acts as a boundary delineating the distribution

range of the Pacific spadenose shark Scoliodon macrorhynchos to the east and, of the Northern Indian

Ocean, S. laticaudus to the west. In addition, the need to determine the species status of Scoliodon

animals from Indonesian waters was identified. The present study reinforced the need to rely on

comprehensive genetic information in addition to external characteristics to assess the species iden‐

tities and distribution range for small sharks and rays that have apparent contiguous coastal distri‐

bution with limited dispersal abilities.

Abstract: Previous examination of the mitochondrial NADH2 gene and morphological characteris‐

tics led to the resurrection of Scoliodon macrorhynchos as a second valid species in the genus, in ad‐

dition to S. laticaudus. This study applied an integrated taxonomic approach to revisit the classifica‐

tion of the genus Scoliodon based on new materials from the Malaysian Peninsula, Malaysian Borneo

and Eastern Bay of Bengal. Mitochondrial DNA data suggested the possibility of three species of

Scoliodon in the Indo‐West Pacific, while the nuclear DNA data showed partially concordant results

with a monophyletic clade of S. macrorhynchos and paraphyletic clades of S. laticaudus and S. cf.

laticaudus from the Malacca Strait. Morphological, meristic and dental characteristics overlapped

between the three putative species. Collective molecular and morphological evidence suggested

that the differences that exist among the non‐sympatric species of Scoliodon are consistent with iso‐

lation by distance, and Scoliodon macrorhynchos remains as a valid species, while S. cf. laticaudus is

assigned as S. laticaudus. The Malacca Strait acts as a spatial delineator in separating the Pacific S.

macrorhynchos (including South China Sea) from the Northern Indian Ocean S. laticaudus. Future

taxonomic work should focus on clarifying the taxonomic status of Scoliodon from the Indonesian

waters.

Keywords: spadenose sharks; integrated taxonomy; synonymy; Indo‐West Pacific; morphometrics;

genetics; distribution range

Citation: Lim, K.C.; White, W.T.;

Then, A.Y.H.; Naylor, G.J.P.;

Arunrugstichai, S.; Loh, K.‐H.

Integrated Taxonomy Revealed

Genetic Differences in

Morphologically Similar and

Non‐Sympatric Scoliodon

macrorhynchos and S. laticaudus.

Animals 2022, 12, 681.

https://doi.org/10.3390/ani12060681

Academic Editor: Martina Francesca

Marongiu

Received: 27 January 2022

Accepted: 5 March 2022

Published: 8 March 2022

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Copyright: © 2022 by the authors. Li‐

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tribution (CC BY) license (https://cre‐

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Animals 2022, 12, 681 2 of 23

1. Introduction

The genus Scoliodon was proposed by Müller and Henle [1] for S. laticaudus Müller

and Henle [2]. Within the family Carcharhinidae, this genus is distinguished from other

genera by its clasper and cranial morphology and very shallowly concave post‐ventral

caudal margin [3]. The genus Scoliodon is morphologically similar to hammerhead sharks

(family Sphyrnidae) in a number of proportional body measurements but is placed in Car‐

charhinidae, as it does not have the laterally expanded head that is characteristic of ham‐

merheads [4]. The genus sits within the subfamily Scoliodontinae and differs from the

other genera within the subfamily, i.e., Rhizoprionodon and Loxodon, by having a greatly

depressed, trowel‐shaped head, broader and more triangular pectoral fins and a more

posteriorly located first dorsal fin (free rear tip about over mid‐bases of the caudal fin) [5].

Scoliodon has long been considered to be a monotypic genus until White et al. [5] res‐

urrected S. macrorhynchos [6] as a second species within the genus. Scoliodon laticaudus is

common along the insular shelf extending from the Northern Indian Ocean to Northeast‐

ern Africa [7]. Scoliodon macrorhynchos is known from Southeast Asia from Taiwan and

China to Indonesia and Sarawak, Malaysia [5]. A possible third species was also reported

from the Bay of Bengal by White et al. [5] and Naylor et al. [8] based on NADH2 sequence

data. These authors suggested that Carcharhias (Physodon) muelleri Müller and Henle [9],

described from Bengal may be an available name for this species, but in the absence of

specimens, this species was not formally resurrected.

The spadenose shark is one of the smallest carcharhinid species, attaining a maxi‐

mum total length of 74 cm [10], occurring in shallow muddy and sandy bottom habitats

[11]. Nearshore elasmobranchs generally have limited dispersal capabilities [4]. For in‐

stance, the bambooshark Chiloscyllium punctatum [12] and the stingray Neotrygon species

[13], both of which are small, show regional population subdivisions with limited genetic

mixing throughout the Indo‐West Pacific. When geographic barriers and the lack of suit‐

able contiguous habitats are combined with a proclivity not to disperse, allopatric specia‐

tion becomes more likely. These factors influenced the redescription of S. macrorhynchos

from the Eastern South China Sea and the suggestion that S. muelleri from the Bay of Ben‐

gal might also be a distinct species [5].

White et al. [5] found that S. macrorhynchos and S. laticaudus showed high intraspecific

variations from morphometric data (as high as 5.2% in some head and snout measure‐

ments) but low interspecific variations; only a limited number of morphometric measure‐

ments differed between the two species, with partly overlapping ranges. For the molecu‐

lar analysis, the interspecific genetic distance of the NADH dehydrogenase subunit 2

(NADH2) gene between S. macrorhynchos and S. laticaudus was about 3%. This degree of

divergence falls at the borderline of “intra” versus “inter”‐specific genetic variations in

sharks and rays. Mobula kuhlii and M. eregoodoo were viewed as one species based on their

close genetic distance (interspecific distance <1.5%) but viewed as distinct species based

on morphological data [14]. Hypanus berthalutzae was viewed as a distinct species from

other closely related Hypanus species based on genetics (interspecific distance 0.82–3.11%),

morphology, and ecological niche modeling data [15]. These examples highlight the chal‐

lenge of distinguishing similar‐looking but potentially distinct species, such as those in

the genus Scoliodon.

Reliance on mitochondrial DNA (mtDNA) alone in elucidating phylogenetic rela‐

tionships among closely related species has been called into question. Reviews by Galtier

et al. [16] and Balloux [17] presented some of the limitations associated with reliance on

mitochondrial data. The concerns raised arose from limited cases of non‐maternally trans‐

mitted mtDNA that may call into question the assumption of reduced within‐individual

diversity [18–20], non‐neutral evolution through selection [21–23], and the nonconstant

mutation rate in mtDNA [24–26]. While these concerns may not necessarily be applicable

in the representation of within‐species history for Scoliodon, the genetic basis for delineat‐

ing S. macrorhynchos as a separate species from S. laticaudus [5] merits a critical review

using more representative sampling with additional markers.

Animals 2022, 12, 681 3 of 23

In this study, both nuclear and mtDNA markers were used in addition to morpho‐

logical data sample specimens across known geographical range of Scoliodon to clarify the

phylogenetic relationships for the group. We included specimens of Scoliodon from the

Malacca Strait, the west coast of Peninsula Malaysia, that had not been previously exam‐

ined. The fine‐scale contemporary distribution range of the Scoliodon genus, especially in

the Indo‐Malaya region, and knowledge gaps were discussed.

2. Materials and Methods

2.1. Sample Collection

Specimens of Scoliodon were acquired at fish landing sites located in the Malacca

Strait on the west coast of Peninsular Malaysia, i.e., Hutan Melintang (3°52’13.6” N

100°55’39.3” E), Sungai Besar (3°40’15.2” N 100°58’52.3” E), and Pasir Penambang

(3°21’03.9” N 101°15’07.0” E), henceforth labeled as S. cf. laticaudus and S. macrorhynchos

from two landing sites in Sarawak in Malaysian Borneo, i.e., Kuching (1°34’04.7” N,

110°22’45.8” E) and Mukah (2°53’50.6” N, 112°05’45.6” E). Tissue samples were taken from

a random subset of specimens (10 each from Malacca Strait and from Sarawak) and stored

in 95% alcohol prior to molecular analyses, while the whole specimens were fixed using

10% formalin and store in 70% alcohol. A subset of specimens, 21 from Malacca Strait and

13 from Sarawak, was preserved whole and retained for subsequent morphological anal‐

ysis by one of us (KCL). Eleven whole specimens of S. cf. laticaudus were also collected

from the Ranong fishing port in Thailand, Eastern Bay of Bengal, during recent surveys

of that landing site [27].

2.2. Molecular Analyses

Two mitochondrial DNA (cytochrome oxidase subunit 1 (COI) and NADH dehydrogenase

subunit 2 (NADH2) regions) were used in molecular species identification and seven nu‐

clear genes following Aschliman et al. [28] DNA (actin‐like protein (ACT), kelch repeat and

BTB domain‐containing protein 2 (KBTBD2), prospero homeobox protein 1 (PROX1), recombina‐

tion activating gene 1 (RAG1), recombination activating gene 2 (RAG2), sec1 family domain‐con‐

taining protein 2 (SCFD2), and transducer of ERBB2.1 (TOB1) region) were used to verify the

taxonomic assignment using mitochondrial DNA. DNA was extracted using 10% Chelex

resin incubated for two minutes at 60 °C, followed by 25 min at 103 °C (modified from

Hyde et al. [29]). Extracted DNA was subjected to Polymerase Chain Reaction (PCR) to

amplify all targeted DNA markers. PCR were carried out either using iTaqTM Plus DNA

Polymerase (iNtRON Biotechnology, INC., Seongnam‐si, Korea) or MyTaqTM Red Mix (Bi‐

oline, London, United Kingdom) in 20 μL of reaction mix containing 2 μL of 10x PCR

buffer; 0.5 μL of dNTP mixture (2.5 mM each); 1 μL of 10‐pmol primer (both primers); 1.25

unit of Taq DNA polymerase; 1 μL of 50‐pg–1.0‐μg DNA templates; and top up with mo‐

lecular‐grade water or 10 μL of MyTaqTM Red Mix premix (mixture of 10x PCR buffer,

dNTPs, and Taq polymerase); 1 μL of 10‐pmol primer (both primers); 1 μL of 50‐pg–1.0‐

μg DNA templates; and top up with molecular‐grade water, respectively. The PCR cycles

for mitochondrial DNA comprised of 2‐min initial denaturation at 94 °C, followed by 30

cycles of 20 s at 94 °C, 20 s at 44 °C (COI) or 52 °C (NADH2), and 1 min at 72 °C and,

subsequently, a final extension of 5 min at 72 °C. The PCR cycles for nuclear DNA com‐

prised 3‐min initial denaturation at 95 °C, followed by 35 cycles of 15 s at 95 °C, 15 s at 52–

60 °C, and 1 min at 72 °C and, subsequently, a final extension of 5 min at 72 °C. Touch‐

down PCR with annealing temperature that decreased 0.3 °C/cycle from 68 °C to 58 °C

was performed on PROX1 due to the amplification of nonspecific DNA at all tested tem‐

peratures between 45 and 60 °C. The primer sets used for all the targeted regions are listed

in Table 1. All PCR products were examined using 1% agarose in TAE buffer prior to the

Sanger sequencing service at Apical Scientific Sdn Bhd (Selangor, Malaysia).

Animals 2022, 12, 681 4 of 23

Table 1. Primers used in this study and their references.

Marker Forward Primer (5′–3′) Reverse Primer (5′–3′) References

COI

FishF2—TCG ACT AAT

CAT AAA GAT ATC GGC

AC

FishR2—ACT TCA GGG

TGA CCG AAG AAT

CAG AA

Ward et al. [30]

NADH2 ILEM—AAG GAG CAG

TTT GAT AGA GT

ASNM—AAC GCT TAG

CTG TTA ATT AA Naylor et al. [31]

ACT ACT‐F—GCT TTC ATC

TCC TTC GGC AGT TTG

ACT‐R—CCA CTG GTA

ATT GGG ATA CTT GGC

Design based on GN’s

sequence of sample

GN1680

KBTBD2

KBT‐F—CTC AGT ATC

TAT CTT CAG TCC TTG

GC

KBT‐R—GCT CTT ACA

CAG GGA TCA GAG

TAG C


sequence of sample

GN1680

PROX1

PRO1‐F—AAT TCT TCA

AGG GAA AGT GCC

CAA G

PRO1‐R—CAG ACT GCT

CCG ACG AGT TTT TG


sequence of sample

GN1680

RAG1

RAG1‐F—CTT ATT CAA

ACC ATC AAC AAC

ACA ACA

RAG1‐R—CTG CAT GAC

TGC TTC CAA CTC ATC


sequence of sample

GN1680

RAG2

RAG2‐F—TCA GAA TCA

AAC AGC CTC ATT TAC

C

RAG2‐R—TTA ATT TCA

TTG GAC CAT TCT GGG

G


sequence of sample

GN1680

SCFD2 SCFD‐F—AGG TGA AAG

CGG TAT TTG TGG TG

SCFD‐R—TGA GCT GCA

GAA CTT CAA ACA

TAG


sequence of sample

GN1680

TOB1

TOB1‐F—ATA TGA AGG

TCA CTG GTA TCC AGA

C

TOB1‐R—GAA AAC

AAA CTC CTT GGC ATT

GGG A


sequence of sample

GN1680

2.3. Phylogenetic Analysis

Sequences were reviewed manually using BioEdit [32], aligned using ClustalX [33],

and finally, trimmed using BioEdit [32]. They were all submitted to the NCBI GenBank

database, with the accession numbers provided in Supplementary Table S1. The following

analyses applied to individual marker, as well as grouped markers by mitochondrial

DNA and nuclear DNA. The aligned sequences were subjected to the best model search

based on Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC)

for Maximum Likelihood (ML) and Bayesian Inference (BI) analyses, respectively, using

Kakusan v.3 [34], as shown in Supplementary Table S2. The generated files were subse‐

quently used for phylogenetic tree construction using Treefinder for ML [35] and MrBayes

for BI [36]. The ML analyses were performed with 1000 bootstrap replicates. The Bayesian

analyses were initiated with a random starting tree and two parallel runs, each of which

consisted of running four chains of Markov chain Monte Carlo (MCMC) iterations for

2,000,000 generations (sampled every 100th generation for each chain). The convergence

and burn‐in from “sump” commands in MrBayes were used to evaluate likelihood values

for post‐analysis trees and parameters. Five thousand trees generated were discarded as

burn‐in (where the likelihood values were stabilized prior before the burn‐in), and the

remaining trees after burn‐in were used to calculate the posterior probabilities using the

“sumt” command.

The finalized ML and BI phylogenetic trees were processed via Figtree v1.3.1

(http://tree.bio.ed.ac.uk/software/figtree/, accessed on 1 October 2014). For mitochondrial

DNA, sequences of closely related species Loxodon macrorhinus and Lamiopsis tephrodes

Animals 2022, 12, 681 5 of 23

were used as outgroups. Sequences of Sphyrna lewini and Rhizoprionodon acutus, on the

other hand, were used as the outgroup for nuclear DNA, as the sequences for Loxodon and

Lamiopsis were not available. As such, the sequence of S. lewini was added to mitochon‐

drial DNA analyses to facilitate the comparison between mitochondrial and nuclear DNA.

Some other sequences available in the National Center for Biotechnology Information

(NCBI) GenBank and Barcode of Life Data (BOLD) systems were also used in the tree

construction for comparison (Supplementary Table S3). Uncorrected p‐distance was cal‐

culated using PAUP* 40b10 software [37] to evaluate the genetic divergence among the

sampled Scoliodon species by sampling areas.

We tested species delimitation using a multispecies coalescent analysis implemented

in ASTRAL 5.7.7 [38,39] and BPP 4.3 [40–42]. In the ASTRAL analysis, two hundred gene

trees were searched under ML + rapid bootstrap for each of the genes using raxmlGUI 1.5

beta [43]. All generated gene trees were combined manually as input into ASTRAL to gen‐

erate a ASTRAL species tree and normalized quartet score. The normalized quartet score

refers to the proportion of gene trees that matched with the species tree; a higher score indi‐

cates greater agreement between gene trees and species tree. In the BPP analysis, we per‐

formed an unguided species delimitation analysis (A11) to test if the Scoliodon species can

be assigned as a single species. We set multiple theta (population size) and tau (divergence

time) combinations using the inverse gamma prior to IG (2, X), with X being 0.1, 0.01, and

0.001. Each analysis was performed twice to confirm the stability of the results.

2.4. Morphological and Meristic Data

Measurement terminology followed Compagno [3,4,44], who assigned names and

abbreviations to measurements often indicated by descriptive phrases (example: snout to

upper caudal origin = precaudal length = PRC). Dentitional terms generally followed

Compagno [3,44,45]. Vertebral terminology, method of counting, and vertebral ratios fol‐

lowed Springer and Garrick [46] and Compagno [3,44,45].

A total of 83 morphometric measurements were obtained from 74 Scoliodon speci‐

mens from a range of locations encompassing a large proportion of the geographic range

of the three ‘species’ types: S. laticaudus, S. cf. laticaudus, and S. macrorhynchos (Figure 1).

A total of 8 specimens of S. laticaudus (India); 32 specimens of S. cf. laticaudus (including

the S. muelleri holotype from ‘Bengal’, Malacca Strait, and the Ranong fishing port in the

Andaman Sea); and 34 specimens of S. macrorhynchos (Hong Kong, Indonesia, Borneo, and

Taiwan) were measured in full (Table 2). Vertebral counts were taken from radiographs

of 13 specimens of S. cf. laticaudus and 13 specimens of S. macrorhynchos. Counts were

obtained separately for the trunk (monospondylous), precaudal (monospondylous and

diplospondylous to the origin of upper lobe of caudal fin), and caudal (centra of the caudal

fin) vertebrae. Tooth row counts were taken in situ or from excised jaws of 7 specimens of

S. laticaudus, 5 specimens of S. cf. laticaudus, and 8 specimens of S. macrorhynchos.

Animals 2022, 12, 681 6 of 23

Figure 1. Lateral view of Scoliodon ‘species’. (a) S. macrorhynchos IPPS WWPLAL#1 (adult male 426

mm TL, fresh), (b) S. cf. laticaudus CSIRO H 8401‐09 (adult male 394 mm TL), and (c) S. laticaudus

MNHN 1123 (female 524 mm TL, preserved).

Table 2. Ranges of proportional dimensions as percentages of the total length for the three ‘species’

of Scoliodon.

S. laticaudus

S. cf. laticau‐

dus

S. macrorhyn‐

chos

n = 8 n = 32 n = 34

Min. Max. Min. Max. Min. Max.

Total length (mm) 169 524 239 490 227 562

Precaudal length 75.6 78.0 75.3 78.0 73.6 78.0

Pre‐second dorsal length 62.6 65.4 62.9 66.7 61.5 66.5

Pre‐first dorsal length 35.1 38.8 33.0 37.7 33.0 38.1

Head length 21.5 29.1 21.5 26.3 21.3 25.6

Head length (horiz) 21.0 28.6 21.0 25.1 20.9 25.0

Pre‐branchial length 17.1 23.5 17.1 20.4 16.5 20.7

Pre‐branchial length (horiz) 16.6 22.6 16.5 19.8 16.0 19.5

Preorbital length 8.9 12.6 8.9 11.7 8.5 11.6

Preorbital length (horiz) 8.1 11.3 7.9 10.8 7.0 10.7

Preoral length 7.1 11.1 7.1 10.4 7.2 10.4

Pre‐narial length 6.6 9.1 6.6 8.7 6.2 8.4

Pre‐narial length (horiz) 5.9 8.2 5.6 8.1 4.8 7.8

Pre‐pectoral length 22.1 26.4 21.5 26.6 20.1 26.2

Animals 2022, 12, 681 7 of 23

Pre‐pelvic length 43.9 48.4 43.9 50.2 43.8 49.2

Snout–vent length 45.9 49.2 45.9 51.4 45.4 50.6

Preanal length 56.7 59.9 56.7 62.0 54.8 60.4

Interdorsal space 16.1 21.7 17.9 21.7 17.9 22.2

Dorsal‐caudal space 7.2 9.3 7.2 9.9 7.2 9.4

Pectoral–pelvic space 16.7 19.7 16.9 20.7 16.8 21.6

Pelvic–anal space 5.2 9.0 5.6 11.1 4.8 8.7

Anal–caudal space 6.4 9.1 6.4 8.8 6.4 9.1

Eye length 1.5 2.2 1.6 2.5 1.3 2.4

Eye height 1.3 2.5 1.3 2.5 1.5 2.2

Interorbital space 7.4 11.2 7.4 9.8 7.5 10.3

Nostril width 1.4 2.0 1.5 2.3 1.4 2.3

Internarial space 4.9 6.9 4.9 6.7 4.9 6.5

Anterior nasal flap length 0.2 0.6 0.2 0.5 0.2 0.6

Mouth length 4.5 5.6 4.1 4.9 3.5 5.2

Mouth width 6.0 7.6 5.3 7.6 5.7 7.6

Upper labial furrow length 0.2 0.6 0.1 0.5 0.1 0.5

Lower labial furrow length 0.8 1.2 0.2 1.5 0.3 1.4

First gill slit height 2.3 3.1 2.3 4.1 2.2 4.0

Second gill slit height 2.3 3.6 2.1 2.6 2.2 3.2

Third gill slit height 2.4 3.8 2.2 4.7 2.3 4.4

Fourth gill slit height 2.4 3.7 2.0 2.8 2.4 3.3

Fifth gill slit height 2.2 3.2 2.1 3.3 2.3 3.1

Intergill length 4.6 5.9 4.6 5.4 4.5 6.4

Head height 6.1 10.2 7.7 9.9 7.0 10.6

Trunk height 7.9 10.8 8.3 10.8 7.8 13.1

Abdomen height 7.5 11.2 10.0 11.4 9.4 13.9

Tail height 6.3 10.2 7.0 9.4 7.5 11.3

Caudal peduncle height 3.9 4.5 3.8 4.5 4.0 5.0

Head width 7.3 9.4 6.9 9.9 7.9 10.8

Trunk width 6.4 8.5 6.5 8.8 6.2 11.8

Abdomen width 5.2 7.1 4.9 6.9 5.2 8.9

Tail width 4.1 5.6 4.2 5.6 4.6 6.5

Caudal peduncle width 1.9 2.7 2.3 3.5 2.2 3.7

Pectoral length 10.2 12.1 9.8 11.6 9.9 11.7

Pectoral anterior margin 9.5 12.1 9.4 11.5 9.2 11.9

Pectoral base 4.5 6.6 5.2 6.4 4.8 6.6

Pectoral height 7.8 10.3 7.4 10.3 7.5 10.1

Pectoral inner margin 5.2 6.2 4.6 6.4 4.3 6.2

Pectoral posterior margin 6.3 10.6 6.8 12.5 6.8 9.8

Pelvic length 7.3 8.7 7.1 8.9 6.9 8.3

Pelvic anterior margin 4.7 5.4 4.3 6.0 4.3 5.6

Pelvic base 4.7 5.6 4.3 6.3 4.3 6.1

Pelvic height 3.2 4.3 2.3 4.4 2.7 4.2

Pelvic inner margin length 2.2 3.7 2.1 3.9 2.2 3.5

Pelvic posterior margin length 3.4 5.3 3.4 5.3 3.8 5.1

Clasper outer length 6.0 9.0 4.5 10.2 4.0 10.0

Animals 2022, 12, 681 8 of 23

Clasper inner length 8.4 11.8 6.4 12.4 6.5 12.1

Clasper base width 1.0 1.4 0.6 1.7 0.6 1.4

First dorsal length 13.3 15.6 13.3 15.7 12.9 15.5

First dorsal anterior margin 11.1 13.5 11.8 14.3 11.2 14.6

First dorsal base 8.9 10.9 8.9 11.4 8.8 11.0

First dorsal height 6.6 8.6 5.8 8.8 6.5 9.0

First dorsal inner margin 3.8 5.1 3.9 5.3 3.5 4.9

First dorsal posterior margin 6.7 9.2 5.7 9.0 6.2 8.9

Second dorsal length 7.5 9.3 7.3 9.1 6.9 8.6

Second dorsal Anterior margin 4.1 5.5 3.4 5.5 3.4 5.0

Second dorsal base 4.0 4.8 3.2 4.9 3.2 4.8

Second dorsal height 1.7 2.2 1.2 2.4 1.3 2.0

Second dorsal inner margin 3.2 4.7 3.8 5.1 3.3 4.8

Second dorsal posterior margin 3.8 5.3 3.9 4.9 3.6 4.7

Anal length 11.4 13.5 9.6 13.7 10.8 14.1

Anal anterior margin 5.1 6.7 4.1 7.0 4.9 7.8

Anal base 8.0 10.3 6.1 10.3 7.2 11.2

Anal height 2.8 3.7 2.2 3.6 2.6 3.8

Anal Inner margin 3.0 3.9 3.0 3.9 2.8 4.1

Anal posterior margin 6.6 8.4 5.8 8.8 6.5 8.9

Dorsal caudal margin 22.0 24.9 21.6 24.6 21.9 25.6

Pre‐ventral caudal margin 8.5 10.2 7.8 10.7 8.0 10.5

Lower post‐ventral caudal margin 3.4 4.7 2.9 5.0 2.9 4.8

Upper post‐ventral caudal margin 9.5 11.5 8.9 11.0 9.1 12.3

Caudal fork width 5.4 7.5 5.4 6.8 5.9 7.1

Caudal fork length 7.8 9.7 8.0 9.8 7.8 9.8

Subterminal caudal margin 3.9 5.6 3.1 4.7 3.1 5.3

Subterminal caudal width 2.6 3.4 2.6 3.5 2.7 3.4

Terminal caudal margin 4.5 7.4 4.8 6.8 4.9 7.3

Terminal caudal lobe 7.6 8.9 6.8 8.6 7.2 9.3

Second dorsal origin 4.6 6.9 3.0 6.9 5.2 9.1

Second dorsal insertion 0.5 2.2 0.6 2.0 0.6 2.7

Mid‐base first dorsal fin to pectoral in‐

sertion 10.9 12.7 10.5 13.4 11.0 14.6

Mid‐base first dorsal fin to pelvic

origin 4.4 6.2 4.6 7.9 4.4 7.6

First dorsal insertion to pelvic mid‐

base 2.8 3.9 2.8 5.4 1.9 5.1

Pelvic mid‐base to second dorsal

origin 12.9 18.1 13.6 18.1 13.5 19.0

2.5. Multivariate Analyses

Morphometric measurements, as % total length (TL), were subjected to nonmetric

multidimensional scaling (MDS) ordination (Primer v7.0 package, Quest Research Lim‐

ited, Auckland, New Zealand) to determine whether significant differences between pu‐

tative species exist or whether intraspecific variations of a single species is a factor. One‐

way Analyses of Similarity (ANOSIM) were employed to test whether morphometric

measurements differed significantly among size classes. Similarity Percentages (SIMPER)

Animals 2022, 12, 681 9 of 23

were employed when a pairwise ANOSIM result was significant at p < 0.05 to determine

what characters contributed most to the observed differences. To determine if significant

differences between size classes exist, samples were allocated to one of four arbitrary size

classes: (1) <249 mm TL, (2) 250–299 mm TL, (3) 300–399 mm TL, and (4) >400 mm TL.

Morphometric measurements were analyzed without transformation since the prelimi‐

nary analyses revealed that the stress levels were acceptable (i.e., <0.3) for MDS analyses

(see Clarke and Gorley [47]). Several measurements, associated with the clasper and trunk

and abdomen heights and widths, were not available for measurement for all individuals,

so these characters were excluded from the MDS analysis.

2.6. Museum Holdings

Collection details for the 74 Scoliodon specimens examined are provided in Supple‐

mentary Data S1. Specimens are referred to by the following prefixes for their registration

numbers: BMNH, British Museum of Natural History, London; IPPS, Sarawak Fisheries

Research Institute, Bintawa, Malaysia; CSIRO, Australian National Fish Collection, Ho‐

bart; RMNH, Rikjsmuseum van Natuurlkjke Histoire, Leiden; and MNHN, Museum Na‐

tional d’Histoire Naturelle, Paris, France.

3. Results

3.1. Molecular Analysis

Using the NADH2 and COI mitochondrial DNA sequences (Figure 2 and Supplemen‐

tary Figure S1a,b), three monophyletic groups with moderate‐to‐full support bootstrap

values (ML 58.3—100/BI 68—100) were identified based on sampling locations, i.e., Scoli‐

odon laticaudus from the Indian Ocean (based on samples from the west coast of India),

Scoliodon macrorhynchos from South China Sea (Kuching and Mukah, both localities in Sa‐

rawak, which were grouped with samples from China and Taiwan), and a possible third

species from the Malacca Strait, tentatively labeled as S. cf. laticaudus, were grouped with

samples from Bangladesh, Myanmar, and Thailand. The uncorrected p‐distances among

these three monophyletic groups ranged from 0.61 to 3.06% for COI, 2.98 to 4.23% for

NADH2, and 2.12 to 3.19% for the combined mitochondrial DNA (Table 3).

Animals 2022, 12, 681 10 of 23

Figure 2. NADH2COI gene mid‐point rooting phylogenetic relationships of Scoliodon ‘species’

(phylogram). The bootstrap values (ML/BI) are shown at branches. Sequence names in bold are from

the present study.

Table 3. Genetic distance range (mean, in percent) among monophyletic groups in mitochondrial

DNA and nuclear DNA phylogenetic trees. Slat—Scoliodon laticaudus, Scflat—S. cf. laticaudus, and

Smac—S. macrorhynchos.

Slat‐Scflat Slat‐Smac Scflat‐Smac

COI 0.82 (0.61–1.53) 2.35 (1.99–2.75) 2.29 (2.14–3.06)

NADH2 3.05 (2.98–3.27) 3.06 (2.98–3.26) 3.64 (3.46–4.23)

Mitochondrial 2.16 (2.12–2.18) 2.82 (2.71–2.89) 3.05 (2.95–3.18)

ACT 0.10 (0.00–0.25) 0.50 (0.50–0.50) 0.50 (0.25–0.74)

KBTBD2 0.00 (0.00–0.00) 0.22 (0.22–0.22) 0.22 (0.22–0.22)

PROX1 0.00 (0.00–0.00) 0.02 (0.00–0.11) 0.02 (0.00–0.11)

RAG1 0.12 (0.12–0.12) 0.12 (0.12–0.12) 0.02 (0.00–0.12)

RAG2 0.54 (0.45–0.61) 0.91 (0.91–0.91) 0.58 (0.45–0.61)

SCFD2 0.13 (0.00–0.21) 0.21 (0.21–0.21) 0.17 (0.00–0.42)

TOB1 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.00)

Nuclear 0.12 (0.10–0.14) 0.25 (0.25–0.25) 0.19 (0.16–0.21)

The estimated trees for Scoliodon species using nuclear DNA (Figures 2 and 3 and

Supplementary Figure S1) showed partial agreement with those using mitochondrial

DNA. Three out of five individual nuclear DNA gene trees indicated monophyly of the

Scoliodon genus (Prox1, RAG1, and TOB1) (Supplementary Figure S1c–i). Topologies of

concatenated nuclear DNA estimated tree showed two monophyletic groups, S.

Animals 2022, 12, 681 11 of 23

macrorhynchos and S. laticaudus–S. cf. laticaudus groups (Figure 3). The uncorrected p‐dis‐

tance for nuclear DNA among the three monophyletic groups identified from mitochon‐

drial DNA ranged from 0 to 0.91% (mean 0.2%) (Table 3).

Figure 3. Nuclear gene mid‐point rooting phylogenetic relationships of Scoliodon ‘species’ (phylo‐

gram). The bootstrap values (ML/BI) are shown at branches.

The species tree estimated in ASTRAL for both mitochondrial and nuclear DNA were

topologically congruent with their respective gene trees and had a normalized quartet

score of 0.81 and 0.61, respectively (Figure 4). The BPP run supported both estimations

from traditional phylogenetic analyses (Bayesian and ML) and ASTRAL. Specifically, the

BPP run on mtDNA supported the separation of Scoliodon into three separate species with

a probability of 1 under any combination of the theta and tau priors. The BPP run on nu‐

clear DNA, on the other hand, varied depending on the theta and tau prior settings; set‐

tings of theta at 0.1 regardless of tau prior supported the monospecificity of Scoliodon

(probability >0.99), theta at 0.001 in combinations with tau at 0.01 and at 0.001 supported

separation into three species (probability 0.65–0.88), and other settings in between sup‐

ported the combination of S. laticaudus and S. cf. laticaudus as a separate group from S.

macrorhynchos (probability 0.51–0.61).

Animals 2022, 12, 681 12 of 23

Figure 4. ASTRAL species tree of Scoliodon species for (a) mtDNA and (b) nuclear DNA.

3.2. Morphology and Meristics

No nonoverlapping morphometric ranges were found between the three putative

‘species’ of Scoliodon. Likewise, vertebral counts strongly overlapped between the three

‘species’. No dental morphological differences were detected between the three Scoliodon

‘species’.

The MDS analysis of the measured Scoliodon specimens showed considerable over‐

laps among the three ‘species’ (Figure 5a). Measurements of the limited S. laticaudus sam‐

ples were highly variable but generally fell within the two overlapping clusters of S.

macrorhynchos and S. cf. laticaudus animals. ANOSIM showed that the ‘species’ were sig‐

nificantly different overall (p < 0.01) although the global R2 value was very low (0.24).

Similarly, pairwise comparisons between the three ‘species’ were also significantly differ‐

ent (p < 0.01) but with low R2 values (0.18–0.42).

When the same ordination plot was coded by size class (1 = <250 mm TL, 2 = 250–299

mm TL, 3 = 300–399 mm TL, and 4 = >400 mm TL), the samples for each size class formed

only partially overlapping groups, with the smallest specimens to the left of the plot and

the largest to the right (Figure 5b). ANOSIM showed that the size classes significantly

different overall (p < 0.01), and with a higher global R2 value (0.54). All pairwise compar‐

isons of size classes were also significantly different (p < 0.01), with generally higher R2

values (0.3–0.96). The measurements shown by SIMPER to be the most responsible for the

differences between the size classes were pre‐anal length, pectoral–pelvic space, pre‐pec‐

toral length, pre‐pelvic length, head length, and pre‐first dorsal length.

Animals 2022, 12, 681 13 of 23

Figure 5. Nonmetric multidimensional scaling (MDS) ordination of Scoliodon ‘species’ morphomet‐

ric percentages (% TL): (a) coded by species and (b) coded by size class.

4. Discussion

Based on a combination of nuclear and mitochondrial markers, the evidence supports

the split proposed by White et al. [5]. Evidence from mtDNA suggests genetic isolation

among the three ‘species’ types; S. laticaudus from India is a separate species from S.

macrorhynchos from Sarawak, Malaysian Borneo that appears to cluster with samples from

China and Taiwan. Evidence from the pooled nuclear markers group S. cf. laticaudus (from

Malacca Strait) with S. laticaudus. Both molecular and morphological data presented sug‐

gest that any differences that exist among the species of Scoliodon are consistent with iso‐

lation by distance. We found no evidence of sympatry among any of the three ‘species’.

Presently, we cautiously recommend that S. cf. laticaudus of the Malacca Strait be assigned

as S. laticaudus. These results and the updated distributional range of the Scoliodon species

are discussed below.

Animals 2022, 12, 681 14 of 23

4.1. Taxonomic Conclusions and Recommendations

The decision to resurrect S. macrorhynchos as distinct from S. laticaudus was primarily

based on the NADH2 sequence data obtained in White et al. [5]. Recent studies have high‐

lighted that the use of single mitochondrial markers alone to distinguish between species

can be questionable, especially in light of discordant species trees using mitochondrial

and nuclear DNA (for example, Chimaera ogilbyi in Finucci et al. [48], freshwater snail ge‐

nus Cipangopaludina in Hirano et al. [49], and terrapins (family Emydidae) in Wiens et al.

[50]). In the case of Scoliodon, there is considerable concordance between mitochondrial

and nuclear signals to support the conclusion of White et al. [5], i.e., the resurrection of S.

macrorhynchos as a valid species and separate from S. laticaudus from India.

Phylogenetic and species trees using combined mitochondrial markers group S.

macrorhynchos from Sarawak Borneo and from China together, but the same cannot be said

for nuclear markers due to the nonavailability of China sequences. Both mitochondrial and

nuclear phylogenetic trees mostly support S. macrorhynchos from Sarawak Borneo as sepa‐

rate from S. cf. laticaudus from the Malacca Strait. The discordance between mitochondrial

and nuclear signals arises regarding the relationship of S. cf. laticaudus and S. laticaudus.

Ambiguity in individual nuclear signals underscores the need to use multiple genes to infer

species relationship, and concatenated nuclear signals provisionally group Scoliodon indi‐

viduals from the Malacca Strait as S. laticaudus. In addition to congruence between mito‐

chondrial and nuclear data, congruence between molecular and morphological characteris‐

tics has also been employed to delimit species (e.g., Finucci et al. [48] and Petean et al. [15]).

For Scoliodon, White et al. [5] documented only mean differences in several morphometric

characteristics but with ranges partially overlapping, i.e., head length, pre‐pectoral length,

lower labial furrow length, and second dorsal fin origin to anal fin origin. The more com‐

prehensive morphological data presented in this study did not find any nonoverlapping

morphological differences in the Scoliodon specimens examined. However, given the high

intraspecific variability in measurements from S. laticaudus (Figure 5a), measurements from

additional individuals across a broad distribution range are important to clarify the mor‐

phological distinctions between S. laticaudus and S. macrorhynchos.

The available molecular evidence delimits the Malacca Strait as the easternmost

boundary for the range of S. laticaudus, thereby extending the distribution of the species

based on the most recent International Union for Conservation of Nature (IUCN) assess‐

ment [11]. The Malay Peninsula appears to serve as a contemporary physical barrier be‐

tween the two species. This pattern has been seen for a number of coastal‐associated spe‐

cies with limited dispersal abilities, such as bamboosharks [12], guitarfishes [51], groupers

[52], sea snails [53], and a number of mangrove species [54]. The molecular differences

between morphologically similar but non‐sympatric S. macrorhynchos and S. laticaudus

suggest a relatively recent divergence due to geographical isolation with limited mixing

that drove allopatric speciation, which is feasible given the complexity of the past geolog‐

ical history of the Sundaland region [55]. Further population genetic studies to corroborate

this will help shed light on the evolutionary history and biogeography of the species.

Another important aspect to investigate for Scoliodon is the population genetic struc‐

ture. Scoliodon is one of the top landed sharks in terms of both abundance and biomass in

surveyed areas within Malaysia [56,57]. A strong coastal affiliation [7] and limited disper‐

sal due to small size are traits that likely promote genetic differentiation and, thus, in‐

crease their vulnerability to localized fishing impacts. A similar pattern of a fine‐scale

population structure has been revealed for a similar small‐sized benthic coastal shark,

Chiloscyllium punctatum, that is subject to high fishing pressure in the Southeast Asian re‐

gion [12,58]. Further investigation into the genetic structure of Scoliodon in Southeast Asia

and Indian waters is warranted given the high fishing pressure exerted [59].

Animals 2022, 12, 681 15 of 23

4.2. Geographic Range

Distributional ranges for species are often based on a combination of literature

sources and expert opinions; therefore, validating some occurrences can be difficult. Since

Scoliodon is herein confirmed with two valid species, notwithstanding the possibility of

another in the Bay of Bengal, it is important to critically investigate the full distributional

range for S. laticaudus and S. macrorhynchos. The identity of Scoliodon at locations without

genetic sequences is putatively assigned as either S. cf. laticaudus or S. cf. macrorhynchos

using the Malay Peninsula as the genus distribution break. The resulting distributional

range is displayed in Figure 6, with questionable occurrences noted. Investigation of the

range is discussed below in an east to west direction.

Figure 6. Map of the Indo‐West Pacific region showing the refined range of Scoliodon species based

on the materials examined and a critical examination of the literature. Dubious range locations are

highlighted with a question mark. Red = S. laticaudus, blue = S. macrorhynchos, and green = Scoliodon

sp. (verification needed).

Off Japan, S. cf. macrorhynchos has been recorded as a rare occurrence from Kochi

Prefecture [60] (as S. sorrakowah). Although listed as occurring off the Pacific coast of

Southern Japan by Nakaya [61] and Nakabo [62], it is noticeably absent from checklists of

coastal fishes in prefectures on the Pacific coast of Southern Japan, e.g., Mie [63], Kago‐

shima [64], and Nagasaki [65]. Furthermore, nine specimens of Scoliodon deposited in Jap‐

anese collections with geographic data were caught in either China, Taiwan, or Vietnam

(accessed via http://science‐net.kahaku.go.jp/ on 28 February 2022). The distribution off

Southern Japan appears to be erroneous and should not be included in the range of this

species. It has not been previously recorded from South Korea, but Cho et al. [66] reported

on a single specimen collected from a Yeosu fish market, Busan in 1995 identified as S.

laticaudus and supposedly caught from the South Sea of Korea. Off China, Wang [67] noted

that S. macrorhynchos was abundant off Wenzhou in Southern Zhejiang Province in late

spring and early summer but rarely caught in the northern part of the province. Zhu et al.

[68] also recorded S. macrorhynchos (identified as S. laticaudus) from Zhejiang Province but

Animals 2022, 12, 681 16 of 23

throughout much of the year. Lam and Sadovy de Micheson [69] found that Scoliodon,

identified as S. laticaudus, was the most abundant shark species present during compre‐

hensive market surveys off the Fujian, Hainan, and Guangdong Provinces of China, as

well as off Hong Kong. Likewise, Ebert et al. [70] noted that this species was very abun‐

dant in fisheries catches around Taiwan.

Naylor et al. [8] provided numerous NADH2 sequences from specimens caught off

Vietnam recorded during local ichthyofaunal surveys. Orlov [71] listed Scoliodon spp. as

one of the pelagic predators found in marine waters off Cambodia, which likely refers

wholly or in part to S. cf. macrorhynchos. Deechum [72] and Springer [73] included records

of Scoliodon (identified as S. laticaudus) from the Gulf of Thailand. No Scoliodon individuals

were recorded during comprehensive ichthyofaunal surveys along the east coast of Pen‐

insular Malaysia ([57] Lim et al., unpublished data) but are caught in high abundance in

the waters of the west coast of Peninsular Malaysia. As verified by Compagno et al. [74],

Scoliodon was largely absent in the Philippines. A recent listing of this species in the Phil‐

ippines elasmobranch identification guide by Alava et al. [75] was likely based on an old

record of misidentified Loxodon or Rhizoprionodon. In Malaysian Borneo, none were rec‐

orded from off Sabah from multiple fish surveys, but S. macrorhynchos is caught in high

abundance off Sarawak ([76] Lim et al., unpublished data, and Manjaji‐Matsumoto pers.

comm.). Scoliodon was not recorded in shark catches off Bintan Island in the Riau Archi‐

pelago of Indonesia just to the southeast of Singapore [77].

In Indonesia, S. cf. macrorhynchos appears to be restricted to Kalimantan [76] and

around the river outflows of Eastern Sumatra that flow into the Malacca Strait [78]. It has

not been recorded in the literature from West Sumatra or from recent landing site surveys

(Fahmi, pers. comm.). Although Bleeker [6] described S. macrorhynchos from a juvenile spec‐

imen from off Batavia (= Jakarta), which would have likely been caught locally, it has not

been recorded off Java in surveys over the last half a century (e.g., Widodo et al. [79] and

Widodo and Mahiswara [80]). Springer [73] also listed a specimen deposited at the Smith‐

sonian Institute (USNM 72479) from Batavia (= Jakarta, West Java). This specimen was col‐

lected by Owen Bryant and William Palmer in 1909 during a natural history specimen col‐

lection trip [81]. Despite being the most abundant species found in recent surveys of the

Muara Baru fishing port in Jakarta [82], these were caught in South Kalimantan and only

landed in Jakarta. Due to the lack of accurate baseline information, it is not possible to de‐

termine whether Scoliodon has been extirpated from Javan waters due to overexploitation.

Arunrugstichai et al. [27] recorded S. laticaudus as one of the most abundant shark

species landed off the Andaman Coast of Thailand. Psomadakis et al. [83] stated that this

species is found in coastal waters and lower reaches of the rivers in Myanmar. Jit et al. [84]

recorded it as the most abundant shark species based on surveys of two landing centers

in Bangladesh, i.e., Chittagong and Cox’s Bazar. Scoliodon laticaudus is abundant off the

Indian coastline, with verified records from all coastal states (from east to west): Andaman

and Nicobar archipelago [85,86], West Bengal [87], Orissa [88,89], Andhra Pradesh [90],

Tamil Nadu [91], Kerala [92], Karnataka [93,94], Goa [95], Maharashtra [96,97], and Guja‐

rat [98]. Scoliodon laticaudus has not been recorded from the Indian union territory of Lak‐

shadweep (formerly Laccadive Archipelago) nor further south in the Maldives or Chagos

Archipelago. The presence of S. laticaudus off Sri Lanka is less clear. Some checklists have

included this species from Sri Lankan waters, e.g., Misra [99] (as Scoliodon sorrakowah),

Mendis [100] (as Carcharias laticaudus), and De Silva [101]. However, recent surveys of 15

fish markets around Sri Lanka recorded no Scoliodon [102]. Likewise, Moron et al. [103]

did not include this species as present off the west coast of Sri Lanka. Given that Scoliodon

is usually found in abundance where it occurs, its absence is notable in these studies. Thus,

it may be absent from Sri Lankan waters or restricted to only the northern part of Sri Lanka

around Palk Bay and the Gulf of Mannar, where it is known to be abundant on the respec‐

tive Indian coastlines. Off Pakistan, S. cf. laticaudus was recorded from the coasts of the

Sindh Province (Misra [104] as S. sorrakowah) and a single specimen recorded during port

surveys at Jiwani in Westernmost Balochistan Province, close to the Iranian border [105].

Animals 2022, 12, 681 17 of 23

The range of Scoliodon has recently included the Persian Gulf and parts of East Africa

[7,10]. Bishop [106] and Sivasubramanian and Ibrahim [107] recorded it from off Kuwait

and Qatar, respectively, but more recent comprehensive surveys of these locations, as well

as of Bahrain and the United Arab Emirates, did not record any S. laticaudus in fisheries

landings [108–110]. Amojil et al. [111] included this species as only possibly occurring in

the Persian Gulf due to the lack of verifiable records. Scoliodon cf. laticaudus was not rec‐

orded during comprehensive surveys of fish landing sites in Oman [112,113]. It was also

not recorded from catches of Russian trawlers operating off the entire Yemen coast (in‐

cluding Socotra Island) between 1985 and 1990 [114] or in a recent comprehensive survey

of the fish fauna of Socotra Islands [115].

Scoliodon cf. laticaudus was included as part of the marine fauna of Somalia [116] and

reported as rare in the Somali shark fishery [117]. Although included in a species catalog

of Kenya [118], surveys of catches in small‐scale fisheries off Kenya over the last decade

have not recorded any individuals of this species ([119] B. Kiilu, pers. comm.). Compagno

[4] included Tanzania in the range for S. cf. laticaudus and also included it as present in

Mozambique [120]. However, this species has not been recorded from fishery bycatches

in recent years in either Mozambique or Tanzania (A. Marshall, S. Pierce, C. Rohner, and

D. Ebert, pers. comm.). The presence of Scoliodon in the fauna of East Africa from Somalia

to Mozambique is dubious. Where S. laticaudus is found, they are typically caught in high

numbers and common in coastal waters. It is more likely that they are misidentifications

of similar species, e.g., Rhizoprionodon acutus, which was previously referred to as Scoliodon

walbeehmi throughout the Indo‐West Pacific before being synonymized. Thus, the East Af‐

rica distribution of S. laticaudus is treated as dubious.

The present distribution delineation is mostly consistent with the recently published

IUCN assessment for S. laticaudus [11] and S. macrorhynchos [121]. In a largely contiguous

coastline distribution of Scoliodon (Figure 6), we noted two contemporary spatial ‘breaks’,

i.e., along the east coast of the Malaysian Peninsula and off the Sabah coastline of North‐

eastern Borneo. These breaks could be due to the presence of unsuitable bottom habitats

for the species (Manjaji‐Matsumoto, pers. comm.) and also reflect the complex evolution‐

ary history of the Sundaland region. Notably, the presence and taxonomic status of Scoli‐

odon in the Indonesian region, especially along Eastern Sumatra and along the Kalimantan

coastline (Figure 6), needs to be investigated using an integrative approach, i.e., molecular

and morphological analyses. It was hypothesized that animals along Eastern Sumatra are

S. laticaudus, while those in Kalimantan waters are S. macrorhynchos, with the Karimata

Strait acting as a physical and/or genetic barrier—this is consistent with evidence pre‐

sented for the genetic structure seen for C. punctatum [12].

5. Conclusions

Collective evidence from mitochondrial DNA, nuclear DNA, and morphological

analyses clearly supports the previous resurrection of S. macrorhynchos as distinct species

from S. laticaudus. Genetic distinctiveness between the two species is likely a product of

isolation by distance with the Malaysian Peninsula acting as a physical barrier. The iden‐

tity of Scoliodon from Indonesian waters remained unverified and should be the focus for

future taxonomic studies. Both Scoliodon species are currently classified as “near threat‐

ened” in the IUCN Red List. With the new evidence from this study, we recommend up‐

dating the distribution information of these species and investigating the taxonomic status

of Scoliodon animals from Indonesian coastal waters.

Supplementary Materials: The following supporting information can be downloaded at

www.mdpi.com/article/10.3390/ani12060681/s1: Table S1: Genetic samples used in this study with

locality data and GenBank accession numbers for each of the mitochondrial and nuclear markers.

Table S2: Best model selected for maximum likelihood and Bayesian inference analysis according to

each marker and the combined markers. Table S3: NCBI GenBank and Barcode of Life Data (BOLD)

Systems accession number of the reference sequences used in the analyses. Data S1: Collection data

Animals 2022, 12, 681 18 of 23

for all specimens of Scoliodon examined in this study. Figure S1a: COI gene phylogenetic relation‐

ships of Scoliodon species (phylogram). The bootstrap values (ML/BI) are shown at branches. Se‐

quence names in bold were from the present study. Figure S1b: NADH2 gene phylogenetic relation‐

ships of Scoliodon species (phylogram). The bootstrap values (ML/BI) are shown at branches. Se‐

quence names in bold were from the present study. Figure S1c: ACT phylogenetic relationships of

Scoliodon species (phylogram). The bootstrap values (ML/BI) are shown at branches. Figure S1d:

KBTBD2 phylogenetic relationships of Scoliodon species (phylogram). The bootstrap values (ML/BI)

are shown at branches. Figure S1e: PROX1 phylogenetic relationships of Scoliodon species (phylo‐

gram). The bootstrap values (ML/BI) are shown at branches. Figure S1f: RAG1 phylogenetic rela‐

tionships of Scoliodon species (phylogram). The bootstrap values (ML/BI) are shown at branches.

Figure S1g: RAG2 phylogenetic relationships of Scoliodon species (phylogram). The bootstrap values

(ML/BI) are shown at branches. Figure S1h: SCFD1 phylogenetic relationships of Scoliodon species

(phylogram). The bootstrap values (ML/BI) are shown at branches. Figure S1i: TOB1 phylogenetic

relationships of Scoliodon species (phylogram). The bootstrap values (ML/BI) are shown at branches.

Author Contributions: Designed the study: K.C.L., W.T.W., A.Y.H.T., and K.‐H.L. Performed the

field work: K.C.L., K.‐H.L., and S.A. Conducted the statistical analysis of the data: K.C.L. and

W.T.W. Conducted the molecular analyses: K.C.L. and G.J.P.N. Captured morphological and me‐

ristic data: W.T.W. and S.A. Wrote the manuscript: K.C.L., W.T.W., and A.Y.H.T. All authors have

read and agreed to the published version of the manuscript.

Funding: Financial support for the lead author K.C.L. was provided by the University Malaya Re‐

search Grant RP018C‐16SUS and the University Malaya Research Fund Assistance BK018‐2015; for

K.‐H.L., funding for rental the facility instruments of molecular analyses in the IOES was provided

by the World Wide Fund‐Malaysia PV049‐2019, the APC was provided by Third Institute of Ocean‐

ography, State Oceanic Administration, China IF004‐2022 and the UM Research Grant RU009H‐

2020; for W.T.W., by the CSIRO National Research Collections Australia; and for G.J.P.N., funding

for some of the molecular analyses was provided through a US National Science Foundation Divi‐

sion of Environmental Biology Award (#1132229). Financial support to collect specimens was pro‐

vided by the Australian Centre for Agricultural Research (Indonesian projects FIS/2000/006 and

FIS/2003/037), National Science Foundation (Borneo projects DEB 0103640 and DEB 0542846), Mur‐

doch University (internal funding for Taiwan collection trip), CSIRO, and the University of Hong

Kong (discretionary funding for guest lecture and collection trip in Hong Kong), Centre for Biodi‐

versity in Peninsular Thailand, and Trocadero group funding (to S.A. for Thailand collection).

Institutional Review Board Statement: The specimens of the spadenose sharks used in this study

were all taken from fish market surveys of Southeast Asia between 2001 and 2016. All specimens

were dead prior to the surveys being undertaken. The fishing port surveys in Thailand were con‐

ducted by SA as part of his Master’s Thesis program through the Prince of Songkhla University, and

permission to collect data was granted in accordance with the Thailand Department of Fisheries.

The fishing port surveys in Malaysia were conducted by KCL as part of his PhD Thesis program

through the University of Malaya. No permission to collect data from Peninsular Malaysia was

needed at the time of the study, and permission to collect data from Sarawak waters was granted

by the Fisheries Research Institute Sarawak, Department of Fisheries Malaysia. All comparative ma‐

terials used in this study were deposited in museum collections around the world and were bor‐

rowed with official loan documentation from these collections. No live animals were collected or

killed during this study. All applicable international, national, and/or institutional guidelines for

the care and use of animals were followed by the authors.

Data Availability Statement: Ranges of the morphological data obtained in this study are provided

in Table 2. The raw morphological data generated during this current study are available from the

corresponding author on reasonable request. All sequences used in this study have been deposited

in GenBank, and the related accession numbers are provided in the related figure and text sections.

Acknowledgments: Thanks to J. Caira (University of Connecticut); K. Jensen (University of Kansas);

P. Last, G. Yearsley, and J. Stevens (CSIRO); Mabel Manjaji‐Matsumoto (Universiti Malaysia Sabah);

Annie Lim (Fisheries Biosecurity Centre Sarawak); Fahmi (Indonesian Institute of Sciences); V. Lam

and S. Lea (University of Hong Kong); and Dharmadi (Research Centre for Capture Fisheries, Ja‐

karta) for their valuable work in the field. We would also like to acknowledge J. Pogonoski (CSIRO)

for capturing the meristic data, H. O’Neill (CSIRO) for providing editorial comments, A. Graham

(CSIRO) for providing collection information and registering specimens, and L. Conboy (CSIRO)

for image preparation. We would also like to thank the following museum staff for allowing

Animals 2022, 12, 681 19 of 23

accessing and assisting with specimen examination: M. van Oijen and R. de Ruiter at the Rijksmu‐

seum van Natuurlijke Histoire (RMNH) in Leiden; R. Causse, B. Séret, G. Duhamel, and P. Pruvost

at the Muséum national d’Histoire naturelle (MNHN) in Paris; and P. Campbell and J. Maclaine at

the British Museum of Natural History (BMNH) in London.

Conflicts of Interest: The authors declare that they have no conflicts of interest.

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